In the field of medical diagnosis, many biomarkers of interest are present in body samples at low concentrations and require label amplifications such as radioluminescence, photoluminescence, gold or polymer microparticles or nanoparticles for detection. With the current medical diagnostic trends of ensuring that most of the population are covered by health screening, reducing medical costs, serving the health-care needs of patients and saving lives in-time, point-of-care systems for medical diagnostics are of great interest. A point-of-care system usually has a disposable microfluidic chip to capture the analytes for on-site detections. In such a system, if fluorescence is used as the detection mechanism for a sandwich assay in which a target antigen is bound between a capture antibody immobilized on a substrate and a detection antibody that is conjugated with a fluorescence dye, the fluorescence signal emitted from the fluorescence dye in such a 2-dimensional substrate is much weaker than the 3-dimensional fluorescence dye emission in homogeneous liquid. Nevertheless, 2-dimensional substrates are attractive candidates for a fully automated point-of-care system as the analytes can be captured on the substrate and detected easily.
In order to compensate the sensitivity loss of the signal due to the 2-dimensional substrate, a well-known method is to utilize surface plasmon resonance (SPR) generated by a gold film to enhance the fluorescence dyes. When a gold film is coated on the glass substrate, high sensitivity enhancement can be achieved by combining a Kretschmann configuration, where SPR excited by the transverse magnetic mode of light on the gold film will enhance the fluorescence signal, and a biomarker can be detected at a limit of detection as low as 2 pg/ml with a photomultiplier tube (PMT) through a dextran matrix on the SPR chip. However, the Kretschmann configuration is a complicated system, not readily portable and expensive due to the need for the photomultiplier.
An alternative to this technology is to utilize the localized surface plasmon resonance (LSPR) generated by gold nanostructures fabricated on glass chip, where the fluorescence can theoretically be enhanced up to 1300 times on the plasmonic hotspots, and at an average of 10-200 times. LSPR can excite the fluorescence dyes at any angle such as normal light incidence, where the light transmission mode or reflection mode can be employed to form a very simple point-of-care system. However, in these experiments, the plasmonic enhancement of the fluorescence dye emission is only feasible to achieve 10 to 100 times sensitivity enhancement, and they require a sensitive photodetector or PMT, or a confocal microscope for detection. The inventors have found that such a system cannot be detected using a dark-field microscope and that there are difficulties in detecting the fluorescence label of the LSPR at a low clinical required sensitivity.
Besides the above mentioned weak signal, organic fluorescence dyes are also disadvantageous due to the small gap between their excitation wavelength and emission wavelength, in which the gap is just 15 to 30 nm. Since the light intensity of the fluorescence emission is much weaker than the excitation, in order to reduce the large background noise due to the crosstalk of the excitation, either a dark-field condenser or a high quality optical filter must be used, both of which are expensive and inconvenient for building up a point-of-care system.
There is a need to provide a method for detecting a target analyte that overcomes, or at least ameliorates, one or more of the disadvantages described above.